Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells

Abstract

Higher eukaryotes must adapt a totipotent genome to specialized cell types with stable but limited functions. One potential mechanism for lineage restriction is changes in chromatin, and differentiation-related chromatin changes have been observed for individual genes. We have taken a genome-wide view of histone H3 lysine 9 dimethylation (H3K9Me2) and find that differentiated tissues show surprisingly large K9-modified regions (up to 4.9 Mb). These regions are highly conserved between human and mouse and are differentiation specific, covering only approximately 4% of the genome in undifferentiated mouse embryonic stem (ES) cells, compared to 31% in differentiated ES cells, approximately 46% in liver and approximately 10% in brain. These modifications require histone methyltransferase G9a and are inversely related to expression of genes within the regions. We term these regions large organized chromatin K9 modifications (LOCKs). LOCKs are substantially lost in cancer cell lines, and they may provide a cell type-heritable mechanism for phenotypic plasticity in development and disease.

Figure 1. LOCK-like clustering of histone H3 lysine-9 dimethylation (H3K9Me2) in human placenta

Freshly purified native chromatin digested with micrococcal nuclease was immunoprecipitated with antibody to H3K9Me2 and hybridized to human genomic tiling arrays (ChIP-on-chip), revealing two LOCK-like clusters of ~2 Mb each in the chromosome 15 Prader-Willi/Angelman syndromes imprinted gene region. The X axis represents genomic positions and the Y axis gives log₂ ratios of ChIP over input signal. The gray dots represent individual probe data, and the red line denotes the smoothed curve by sliding windows. The orange bars in the middle show locations of known CTCF binding sites and gene annotations are given on the bottom. The paternally expressed genes (Mkrn3, Ndn, Snrpn and Gabrb3) are localized within and near the edges of LOCK, whereas the maternally expressed genes (Ube3a and Atp10a) are localized between the LOCKs. CTCF binding sites are largely located at the boundaries of the LOCKs.

Figure 2. H3K9Me2 LOCKs are conserved between human and mouse

ChIP-on-chip data of the ENCODE region ENr213 in human placenta and its syntenic region in mouse liver. Dots and curves are as in Fig. 1. The location of H3K9Me2 LOCKs is highly consistent between human and mouse. The LOCK on the right part of the figure begins at the 3′end of the CDH2 gene in both human and mouse. See also Supplementary Fig. 4.

Figure 3. H3K9Me2 LOCKs arise during differentiation

a, Depicted is a summary of data from a 10 Mb region of mouse chromosome 8. Locations of LOCKs in undifferentiated mouse embryonic stem (ES) cells, differentiated ES cells, liver and brain are denoted by green, red, orange and blue bars, respectively. b, LOCKs are almost completely absent in differentiated G9a knockout ES cells, compared to wild type ES cells. The black and red lines represent smoothed curves of H3K9Me2 in differentiated WT and G9a knockout ES cells, respectively. See also Supplementary Fig 6.

Figure 4. Tissue-specific H3K9Me2 LOCKs are associated with differential gene expression in liver and brain

Levels of gene expression in liver (X axis) and brain (Y axis) of genes in liver LOCKs only (a), genes in brain LOCKs only (b), genes in LOCKs common to liver and brain (c) and genes not in LOCKs in liver or brain (d). Genes that lay within LOCKs in the liver but not in the brain were largely silenced in liver, but showed a broad range of expression in brain (P = 0.002). Similarly, genes in LOCKs in brain but not liver, while fewer in number were largely silenced in brain, but showed a broad range of expression in liver (P = 10⁻⁹). Genes that lay within LOCKs in both tissues were silenced in both, while genes not in LOCKs in either liver or brain were broadly expressed in both tissues (P = 0.0002 and 0.005 for liver and brain, respectively). e, an example of a gene clusters in a tissue-specific LOCK. Expression levels of transcripts are based on the GNF Atlas track from UCSC genome browser. This region is marked by brain LOCKs, and correspondingly, the genes under LOCKs are silenced (green) in brain but highly expressed (red) in liver.

Figure 5. A model of epigenetic memory of cell type-specific higher-order chromatin structure mediated by H3K9Me2 LOCKs

In undifferentiated ES cells (upper left), the genome is largely unmarked by H3K9Me2 LOCKs, chromosomal positioning is relatively apart from the nuclear membrane and shows global expression. In differentiated cells such as brain (upper right) and liver (lower left), large fractions of the genome are marked by H3K9Me2 LOCKs in a tissue-specific manner. The chromosome may be repositioned through the association of H3K9Me2 and nuclear lamina through unknown mediator proteins, forming lineage-specific nuclear architectures. During cell division, the lamina-LOCK association can be remembered in mitosis (lower right) and the nuclear architecture reestablished through a mechanism dependent on nuclear envelope breakdown and reformation, and enhanced by contiguous sites of H3K9Me2 modification.